The transition state for folding of SEE COMMENTARY an outer membrane protein Gerard H. M. Huysmansa,b, Stephen A. Baldwina,b, David J. Brockwella,c,1, and Sheena E. Radforda,c,1 aAstbury Centre for Structural Molecular Biology, bInstitute of Membrane and Systems Biology, and cInstitute of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom Edited by Alan R Fersht, Medical Research Council Centre, University of Cambridge, Cambridge, United Kingdom, and approved November 18, 2009 (received for review October 15, 2009). Inspired by the seminal work of Anfinsen, investigations of the ever, a detailed structural description of the folding transition folding of small water-soluble proteins have culminated in detailed state of any β-barrel membrane protein remained to be achieved. insights into how these molecules attain and stabilize their native Here we have exploited the experimental amenability of the folds. In contrast, despite theiroverwhelming importance in biology, Escherichia coli outer membrane protein PhoPQ-activated gene progress in understanding the folding and stability of membrane P (PagP) to provide a detailed analysis of the folding mechanism proteins remains relatively limited. Here we use mutational analysis of a β-barrel transmembrane protein. PagP comprises a mono- to describe the transition state involved in the reversible folding meric eight-stranded β-barrel with an N-terminal, amphiphathic of the β-barrel membrane protein PhoPQ-activated gene P (PagP) α-helix (19, 20) (Fig. 1A). The protein functions as an enzyme as from a highly disordered state in 10 M urea to a native protein part of the bacterial stress response mechanism, reinforcing the embedded in a lipid bilayer. Analysis of the equilibrium stability outer membrane by transferring a palmitoyl chain from phospho- and unfolding kinetics of 19 variants that span all eight β-strands lipids to lipopolysaccharides (21). We have previously shown that of this 163-residue protein revealed that the transition-state struc- PagP folds spontaneously into lipid bilayers, commencing from a ture is a highly polarized, partly formed β-barrel. The results provide highly denatured state that lacks residual secondary structure in unique and detailed insights into the transition-state structure for 6 M guanidinium chloride (Gdn-HCl). Having established condi- β-barrel membrane protein folding into a lipid bilayer and are tions under which this transition is fully reversible, we here pre- sent an integrated thermodynamic and kinetic analysis of the consistent with a model for outer membrane protein folding via a folding and unfolding transition of the wild-type protein. Com- tilted insertion mechanism. bined with analysis of the stability and folding properties of 19 ∣ ∣ ∣ ∣ variants of the protein that contain single-point mutations span- beta barrel membrane protein PagP phi-value analysis ning all eight β-strands of the native β-barrel, we describe the protein folding transition state for folding of a β-barrel membrane protein into a lipid bilayer in residue-specific detail. The results reveal a tran- embrane proteins are encoded by up to 30% of genes in sition-state structure that is highly polarized, involving at least Mmost organisms (1) and are crucial to a wide diversity of partial structure formation in all eight β-strands of the native BIOPHYSICS AND essential activities in biology (for example, ref. 2 and references β-barrel, and suggest a route by which a folding polypeptide chain COMPUTATIONAL BIOLOGY therein). Identifying the forces that govern the folding and can both enter and traverse a bilayer to adopt its native state. stability of membrane proteins is thus fundamental to under- standing their functions and elucidating how sequence changes Results give rise to altered functional properties and, for some such Thermodynamic and Kinetic Measurements of the Folding and Unfold- proteins, grave disease (3). Membrane proteins divide into two ing Transition of Wild-Type PagP. In previous work we have shown major classes: proteins that are α-helical in their transmembrane that PagP folds spontaneously from a completely unfolded con- domains and are ubiquitously distributed and proteins that span formation in 6 M Gdn-HCl into di-lauroyl-phosphatidylcholine the membrane in the form of a β-barrel and are found within (diC12∶0PC)-liposomes at a lipid-to-protein ratio (LPR) of 800∶ the outer membranes of Gram-negative bacteria, chloroplasts, 1 in the presence of 7 M urea (22). Here, using tryptophan and mitochondria. α-Helical membrane proteins are difficult to (Trp)-fluorescence as a probe for correct folding, we established unfold to an unstructured state (4), hampering detailed investi- that 0.4 μM PagP denatured in 10 M urea folds efficiently to its gations of their folding mechanisms into the lipid bilayer (5). native state in 100 nm diC12∶0PC-liposomes at an LPR of 3200∶1 in Important progress in folding studies on this class of membrane a transition that is completely reversible and is independent proteins has been made recently, however, using detergent- of both the protein and lipid concentration under the conditions solubilized proteins (6). In particular, partial denaturation of employed (Fig. 1B)(SI Text and Fig. S1). The resulting equili- bacteriorhodopsin followed by refolding/unfolding into mixed brium curve fitted well to a two-state transition from which detergent/lipid micelles by titration of the destabilizing detergent the thermodynamic properties of the protein were determined, ΔG0 60 17 Æ 1 80 ∕ sodium dodecyl sulfate has provided insights into the packing with a free energy for unfolding, UN,of . kJ mol M 6 86 Æ 0 20 −1 −1 of preformed helices around a critically maintained helical core and an UN of . kJ · mol ·M , the latter being a in the denatured state of the protein (7–10). Such an approach cannot be employed, however, to investigate folding of mem- Author contributions: G.H.M.H., S.A.B., D.J.B., and S.E.R. designed research; G.H.M.H. brane proteins into a lipid bilayer, the physicochemical properties performed research and analyzed data; and G.H.M.H., S.A.B., D.J.B., and S.E.R. wrote of which are known to have profound effects on membrane pro- the paper. tein stability and assembly (10–14). By contrast with the recalci- The authors declare no conflict of interest. trance of α-helical membrane proteins to complete denaturation, This article is a PNAS Direct Submission. β -barrel transmembrane proteins are readily unfolded using Freely available online through the PNAS open access option. – membrane-compatible chaotropes (14 16). For outer membrane See Commentary on page 3947. protein A (OmpA), this has allowed energetic contributions of 1To whom correspondence may be addressed. E-mail: [email protected] or functionally important hydrogen bonds (17) and of residues in the [email protected]. anchoring aromatic girdles (18) on stability to be quantified. De- This article contains supporting information online at www.pnas.org/cgi/content/full/ spite their experimental tractability and ubiquity in nature, how- 0911904107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0911904107 PNAS ∣ March 2, 2010 ∣ vol. 107 ∣ no. 9 ∣ 4099–4104 Downloaded by guest on September 24, 2021 Fig. 2. (A) Folding and (B) unfolding kinetics of 0.4 μM PagP in diC12∶0PC- Fig. 1. (A) Cartoon representation of PagP [Protein Data Bank (PDB) ID code liposomes (LPR 3200∶1) measured by Trp-fluorescence. The arrows indicate 1THQ (19); W. L. DeLano, http://www.pymol.org (2002)]; β-strands are labeled increasing urea concentrations (7.8 to 8.8 M and 9 to 10 M urea in steps A–H; periplasmic turns t1–3. (B) Equilibrium refolding (▪) and unfolding (□)of of 0.2 M for folding and unfolding, respectively). (C) Urea-concentration wild-type PagP. The unfolding transitions of PagP variants bearing mutations dependence of the rate constants of folding (▪) and unfolding (□). Lines in residues in (B) the hydrophobic surface, (C) the aromatic girdles, and represent linear fits to each dataset. (D) Urea-concentration dependence (D) the barrel interior are also shown. For these variants residues mutated of the unfolding rate constant of PagP from diC12∶0PC-liposomes measured that are located in the N-terminal half of the protein sequence are indicated using Trp-fluorescence [0.4 μM PagP; LPR 3200∶1 (□)or800∶1 (Δ)] and using by closed symbols, whereas residues in the C-terminal half of the protein CD-spectroscopy (5 μM PagP; LPR 800∶1) at 232 nm (×) and 218 nm (+). Inset, sequence are shown as open symbols. Solid lines are global fits to a two-state CD-spectra of native (N) and unfolded, membrane-associated (U) PagP M ¼ 6 86 Æ 0 20 ∕ ∕ ¼ ½Θ × 103 2 −1 mechanism yielding a common UN . kJ mol M. All experi- [CD-signal mean residue (deg cm dmol )]. ments were performed using 0.4 μM PagP in diC12∶0PC-liposomes at an 3200∶1 LPR of in 50 mM sodium phosphate buffer pH 8 at 25 °C. cur by a reversible two-state reaction at the urea concentrations used (see Materials and Methods and ref. 23). An important prop- measure for the buried surface area in the native structure erty of the transition-state ensemble is how much surface area is (Fig. 1B and Table S1). Refolding experiments using lipids within buried in this species compared with the solvent accessibility of the gel phase at the temperature used (25 °C with diC16∶0PC-li- the native state. This can be calculated from the βT -value, which m ∕M Materials and Methods β posomes or 10 °C with diC14∶0PC-liposomes) indicated that the equals f UN (see and ref. 24). The T folding reaction commences from a highly unfolded state in value of the transition state of PagP of approximately 0.6 thus 10 M urea that is lipid-associated and lacks β-sheet secondary reveals a transition state for the folding of PagP that is placed structure (SI Text and Fig.